Marie Chalons-Cottavoz, Mehdi Lahmar, Klaus Schwamborn, Nicola Beltraminelli, Stephanie Fallot, and Pierre Garrone
- Blink Biomedical SAS [FR/FR]; Gerland Plaza Techsud, 70 Rue Saint-Jean-de-Dieu, 69007 Lyon (FR)
Humanized, mouse or chimerica anti-CD47 monoclonal antibodies are provided. The antibodies bind to human glycosylated and deglycosylated CD47 with an optimized Koff value, they disrupt the human CD47-SIRPα interaction, and find use in various therapeutic, preventative or diagnostic methods. The invention includes isolated antibodies and derivatives and fragments thereof, pharmaceutical formulations comprising one or more humanized or chimeric anti-CD47 monoclonal antibodies; and cell lines that produce these monoclonal antibodies. Also provided are amino acid and nucleotide sequences of the antibodies.
CovalX Technology Used
The epitope that mouse anti-CD47 candidates 20 and 22 recognizes on the hCD47 antigen was determined using a high resolution method. The anti-CD47 antibodies were complexed using a solubilized version of human CD47 extracellular domain which contained a 6His-tag. The resulting anti-CD47 antibody/hCD47 complexes were incubated with deuterated cross-linkers and then exposed to multi-enzymatic cleavage before being analyzed by high resolution mass spectrometry on a machine that had been equipped with a CovalX HM4. From this analysis, it was found that the epitope on hCD47 that is recognized by chimeric candidate 20 is discontinuous and made up of multiple amino acid residues (K59, R63, Y66, T67, H108, T109, T117, and T120). It was also found that the epitope recognized by AB06.12 is different than candidate 20 or 22.
The MALDI analysis of the samples showed the presence of monomeric, dimeric and multimeric organizations of hCD47 occurring naturally in vitro. Thus, the recognition of the dimer of hCD47 by candidate 20 or 22 was further investigated through the use of cross-linking. The anti-CD47 antibody/soluble hCD47 complex was cross-linked by adding the CovalX K200 Stabilization Kit and analyzed again. From this analysis, it was found that candidate 20 binds to the dimer and the monomer of hCD47. It was also hypothesized that candidate 22 may have cross-linked with the hCD47 dimer and monomer. Both candidates were found to bind the monomer and the dimer after purification by SEC-HPLC.
In order to determine whether or not therapeutic antibodies depend on the CD47 glycosylation level for their binding to the target, soluble hCD47 was treated with PNGase. Candidates 20 or 22 were cross-linked using DSS and the CovalX K200 Stabilization Kit before being incubated at room temperature for 180 minutes in order to create anti-CD47 antibody/deglycosylated-hCD47 cross-linked complexes. The samples were then subjected to MALDI analysis to identify peaks. The peak that corresponds to the uncomplexed deglycosylated CD47 was unidentified, thus, leading researchers to believe it was complexed with either of the candidates and further strengthening the belief that antibodies 20 and 22 can identify both glycosylated and N-deglycosylated forms of hCD47.